FAQ: Why don’t I see the expected band from my repaired template?

There are a number of possible issues. First, if possible, it is a good idea to make sure the PCR reaction is properly optimized using a fresh DNA template. For example, if you are amplifying DNA from degraded moth samples, check the PCR reaction using a fresh DNA sample from the same species. Second, there may be PCR inhibitors present in the extracted DNA. This can be tested using a PCR reaction that is known to work and then adding in an aliquot of the extracted DNA. If the PCR reaction is inhibited by the presence of the extracted DNA in question PCR inhibitors may be an issue. In this case be sure to use the supplied BSA in the repair and amplification reactions because BSA is known to mitigate the effects of some types of PCR inhibitors. Another simple, but effective method is to run a series of PCR reactions in which the extracted DNA template is present at a range of dilutions. The idea is to find the point at which the inhibitor is dilute enough to allow PCR and there is still enough DNA template to allow amplification.

Other problems are that the DNA is simply too degraded to be recovered or that the damage present is not repaired by the PreCR Repair Mix, i.e. DNA-protein crosslinks.

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